The present invention relates to novel parasitic helminth phospholipase A2-like (i.e., PLA2) proteins, nucleic acid molecules encoding such proteins and antibodies raised against such proteins. The present invention also includes a method to obtain such nucleic acid molecules, proteins and antibodies as well as use of such compounds to protect animals from infections caused by parasitic helminths. The present invention particularly relates to certain Dirofilaria immitis, Onchocerca volvulus, and Brugia malayi PLA2 nucleic acid molecules, proteins and antibodies as well as their use to protect animals from parasitic helminth infection.
Parasitic helminth infections in animals, including humans, are typically treated by chemical drugs, because there are essentially no efficacious vaccines available. One disadvantage with chemical drugs is that they must be administered often. For example, dogs susceptible to heartworm are typically treated monthly to maintain protective drug levels. Repeated administration of drugs to treat parasitic helminth infections, however, often leads to the development of resistant helminth strains that no longer respond to treatment. Furthermore, many of the chemical drugs cause harmful side effects in the animals being treated, and as larger doses become required due to the build up of resistance, the side effects become even greater. Moreover, a number of drugs only treat symptoms of a parasitic disease but are unable to prevent infection by the parasitic helminth.
It is particularly difficult to develop vaccines against parasitic helminth infections both because of the complexity of the parasite""s life cycle and because, while administration of parasites or parasite antigens can lead to the production of a significant antibody response, the immune response is typically not sufficient to protect the animal against infection.
As an example of the complexity of parasitic helminths, the life cycle of D. immitis, the helminth that causes heartworm, includes a variety of life forms, each of which presents different targets, and challenges, for immunization. Adult forms of the parasite are quite large and preferentially inhabit the heart and pulmonary arteries of an animal. Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system of the dog. One method of demonstrating infection in the dog is to detect the circulating microfilariae.
If the dog is maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by the female mosquito during blood feeding on an infected dog, subsequent development of the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) which can then be transmitted back to the dog through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood. xe2x80x9cOccultxe2x80x9d infection with heartworm in dogs is defined as that wherein no microfilariae can be detected, but the existence of the adult heartworms can be determined through thoracic examination.
Heartworm not only is a major problem in dogs, which typically cannot even develop immunity upon infection (i.e., dogs can become reinfected even after being cured by chemotherapy), but is also becoming increasingly widespread in other companion animals, such as cats and ferrets. Heartworm infections have also been reported in humans. Other parasitic helminthic infections are also widespread, and all require better treatment, including a preventative vaccine program. O. volvulus, for example, causes onchocerciasis (also known as river blindness) in humans. Up to 50 million people throughout the world are reported to be infected with O. volvulus, with over a million being blinded due to infection. Brugia filariids can infect humans and other animals, causing diseases including filariasis (including lymphatic filariasis), elephantiasis and tropical eosinophilia.
Although many investigators have tried to develop vaccines based on specific antigens, it is well understood that the ability of an antigen to stimulate antibody production does not necessarily correlate with the ability of the antigen to stimulate an immune response capable of protecting an animal from infection, particularly in the case of parasitic helminths. A large number of materials are immunogenic and produce sera which test positive in immunoassays for ability to react with the immunizing antigen, but which fail to protect the hosts against infection. Accordingly, the use of serum simply resulting from immunization or from infection by a parasitic helminth to screen for candidate vaccines does not provide sufficient specificity to identify protective immunogens. On the other hand, serum or other components of blood from immunized animals which is demonstrably protective against infection would contain antibodies, cells, or other factors that could selectively bind to potential antigens that, if used as therapeutic compositions, would elicit immune responses that protect against challenge. A method to use serum from immune animals to identify candidate parasitic helminth vaccines is disclosed in U.S. patent application Ser. No. 08/101,283, ibid., also published as PCT International Publication No. WO 92/13560, by Grieve et al., on Aug. 20, 1992.
An alternative approach to finding a suitable parasitic helminth vaccine has been to attempt to identify prominent antigens in the infective stage of the helminth. Researchers have identified several proteins in the infective stage of D. immitis., including, for example, a 35-kilodalton (kD) major surface antigen of D. immitis third stage larvae (Philipp, et al., 1986, J. Immunol. 136, 2621-2627; Ibrahim, et al., 1989, Parasitol. 99, 89-97; Scott, et al, 1990, Acta Tropica 47, 339-353) as well as three major surface proteins of the L4 having molecular weights of 150 kD, 52 kD, and 25 kD (Davis, et al., 1988, Abstract 404, 37th Annual Meeting, Am. Soc. Trop. Med. Hyg.). Scott et al., ibid., also identified a number of other proteins on the surface of D. immitis having molecular weights ranging from 3 kD to 66 kD. None of these proteins has yet been shown to be an effective vaccine.
Furthermore, although several Onchocerca genes have been isolated, genes encoding antigens targeted specifically to L3 and L4 stage larvae have apparently not been reported. In particular, genes encoding antigens that selectively bind to serum obtained from a host that is immune to Onchocerca infection (e.g., O. volvulus infection), apparently have not been isolated, nor apparently have such antigens been characterized.
As such, there remains a need to identify an efficacious composition that protects animals against diseases caused by parasitic helminths and that, preferably, also protects animals from infection by such helminths.
The present invention relates to parasitic helminth PLA2 proteins; to parasitic helminth PLA2 nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins (anti-parasitic helminth PLA2 antibodies), and to inhibitors of phospholipase A2 activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies and inhibitors. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitors, as well as use of such therapeutic compositions to protect animals from diseases caused by parasitic helminths.
One embodiment of the present invention is an isolated parasitic helminth nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dirofilaria immitis PLA2 gene, an Onchocerca volvulus PLA2 gene, and/or a Brugia malayi PLA2 gene. A D. immitis PLA2 gene preferably includes nucleic acid SEQ ID NO:1; an O. volvulus PLA2 gene preferably includes nucleic acid sequence SEQ ID NO:8; and a B. malayi PLA2 gene preferably includes nucleic acid sequence SEQ ID NO:15 and/or SEQ ID NO:18. A PLA2 nucleic acid molecule of the present invention can include a regulatory region of a parasitic helminth PLA2 gene and/or can encode a parasitic helminth PLA2 protein. Particularly preferred PLA2 nucleic acid molecules include nucleic acid sequence SEQ ID NO:1, nucleic acid sequence SEQ ID NO:3, nucleic acid sequence SEQ ID NO:4, nucleic acid sequence SEQ ID NO:6, nucleic acid sequence SEQ ID NO:8, nucleic acid sequence SEQ ID NO:10, nucleic acid sequence SEQ ID NO:11, nucleic acid sequence SEQ ID NO:13, nucleic acid sequence SEQ ID NO:15, nucleic acid sequence SEQ ID NO:16, nucleic acid sequence SEQ ID NO:18, and/or nucleic acid sequence SEQ ID NO:19, as well as allelic variants of one or more of those nucleic acid molecules.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include parasitic helminth PLA2 nucleic acid molecules of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention includes an isolated parasitic helminth PLA2 protein, including a protein that includes a parasitic helminth PLA2 protein. A preferred parasitic helminth PLA2 protein, when administered to an animal, is capable of eliciting an immune response against a natural parasitic helminth PLA2 protein and/or of selectively binding to immune serum derives from an animal that is immune to infection by the parasitic helminth. A preferred parasitic helminth PLA2 protein is a third stage or fourth stage larval protein. In one embodiment, a preferred PLA2 protein has a molecular weight of about 22 kD or of about 20.5 kD as determined by Tris-glycine SDS PAGE. Particularly preferred PLA2 proteins are proteins that include amino acid sequence SEQ,ID NO:2, amino acid sequence SEQ ID NO:5, amino acid sequence SEQ ID NO:7, amino acid sequence SEQ ID NO:9, amino acid sequence SEQ ID NO:12, amino acid sequence SEQ ID NO:14, amino acid sequence SEQ ID NO:17, and/or amino acid sequence SEQ ID NO:20, as well as proteins that are encoded by nucleic acid molecules that are allelic variants of the nucleic acid molecules that encode proteins having any of those SEQ ID NO""s.
The present invention also relates to mimetopes of parasitic helminth PLA2 proteins as well as to isolated antibodies that selectively bind to parasitic helminth PLA2 proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Yet another embodiment of the present invention is a therapeutic composition that is capable of protecting an animal from disease caused by a parasitic helminth. Such a therapeutic composition includes one or more of the following protective compounds: an isolated parasitic helminth PLA2 protein or a mimetope thereof; an isolated parasitic helminth nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dirofilaria immitis PLA2 gene, an Onchocerca volvulus PLA2 gene, and/or a Brugia malayi PLA2 gene; an isolated antibody that selectively binds to a parasitic helminth PLA2 protein; and an inhibitor of phospholipase A2 activity identified by its ability to inhibit parasitic helminth phospholipase A2 activity. A preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Preferred PLA2 nucleic acid molecule compounds of the present invention include naked nucleic acid vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal from disease caused by a parasitic helminth. The method includes the step of administering to the animal a therapeutic composition of the present invention.
Suitable parasitic helminths to use in the production (e.g., recombinant, natural, or synthetic production) of nucleic acid molecules, proteins and antibodies of the present invention include nematodes, cestodes and trematodes, with nematodes (such as filariid, ascarid, strongyle and trichostrongyle nematodes) being preferred, with filariids being more preferred, and with D. immitis, O. volvulus, and B. malayi being even more preferred.
Suitable and preferred parasitic helminths from which to protect animals are as disclosed for use in the production of nucleic acid molecules, proteins and antibodies of the present invention. As such, preferred diseases from which to protect animals include diseases caused by nematodes, cestodes and/or trematodes, with diseases caused by nematodes being more preferred targets, and with diseases caused by filariids being even more preferred targets. Particularly preferred diseases from which to protect animals include heartworm, onchocerciasis and filariasis.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting phospholipase A2 activity of a parasitic helminth. The method includes the steps of: (a) contacting an isolated parasitic helminth PLA2 protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has phospholipase A2 activity; and (b) determining if the putative inhibitory compound inhibits the phospholipase A2 activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting phospholipase A2 activity of a parasitic helminth. Such a test kit includes an isolated parasitic helminth PLA2 protein having phospholipase A2 activity and a means for determining the extent of inhibition of that activity in the presence of a putative inhibitory compound.